Not only do muscles control motor function, but also play an essential role as the largest insulin target tissue in homeostatic maintenance of blood sugar. In type II diabetes, which is explosively increasing at present, “sugar uptake” into muscles that is dependent on insulin stimulation is significantly waning. Moreover, it is known that suitable motor stimulation effectively improves the clinical condition, and exercise therapy is carried out at clinical sites; however, details of the molecular mechanism are unknown.
Muscular motion, in other words contraction and extension of muscle cells, is associated with a large energy consumption as well as mechanical stimulation. Moreover, the multiple stimulation of both creates “healthy muscle cells” with high glucose metabolic capacity within a living body, and maintains proper insulin responsiveness. However, in a culture system, it is difficult to maintain contractional activity of muscle cells that is sufficiently active, and excellent cultured muscle cells that simulate a living body, particularly ones suitable for research into metabolic capacity, do not exist. Thus, efficacy of a drug for muscle cells, etc., has been evaluated using (1) an animal experiment or muscular tissues collected from animals, or (2) an undeveloped cultured muscle cell strain.
Muscular tissues bring out high metabolic capacity through exercise, hence procedures such as forcing animals to run on a treadmill or swim in a tank for a long time were mainly carried out to evaluate the efficacy of a drug that affects the metabolic capacity of muscle cells when muscular tissue collected from the animal was used. Moreover, electrodes were connected to a muscular tissue collected from an experimental animal to activate contraction with an electric pulse in order to prepare a sample. These animal experiments involve ethical issues, in addition to being unsuitable for screening a number of drugs.
On the other hand, an excellent cultured cell system that simulates a living body helps to screen drugs to a large extent. However, a cultured muscle cell system taking into consideration the characteristics of muscle cells, mainly contraction and extension, is poor, and contractile performance of cultured muscle cells prepared under normal culture conditions has hardly been developed. Therefore, they were not developed enough to have high metabolic capacity as described in Biochem. Pharmacol., 2003, Vol. 65, page 249-257, and Am. J. Physiol. (Endoclinol Metab), 2002, Vol. 283, page E514-524, and they did not simulate the muscles of a living body, making them completely unsuitable for research on metabolic capacity.
Furthermore, in Japanese published unexamined application No. 2005-27501, a method of preparing muscle cells having automatic contractile capacity by applying an electric pulse by wave pulse to myoblast cells is described. Moreover, in Japanese published unexamined application No. 2003-225, a cell culture apparatus that passes a vertical electric current and a cell culture method are described.
However, even with the cell culture methods described in these patents, the preparation of muscle cells suitable for research on metabolic capacity such as uptake measurement, etc., has not been successful. Moreover, the prior measurement conditions of metabolic capacity were not suitable for cultured muscle cells.
As described above, energy necessary for life support and movement of muscles is mostly produced by metabolizing sugar responding to various extraneous stimulations such as insulin, exercise, etc., and incorporated within the cells. Such sugar is transported to cells through a facilitated diffusion glucose (glucose transporter; GLUT) family, of which 13 have been identified so far.
Particularly, increased sugar uptake induced by insulin is physiologically important, and, for example, a large part (70-85%) of blood sugar that increases after a meal is incorporated in muscular tissues by insulin stimulation, and hence, muscular tissues play a very important role in controlling the blood sugar of an entire living body.
Therefore, accurately measuring insulin-dependent sugar uptake activity within muscle cells is essential to applications in a wide variety of industries such as developing drugs that increase insulin sensitivity, finding causes that decrease insulin sensitivity, and providing diagnoses at an early stage of diabetes, etc., as well as applications to the field of basic research.
In muscular tissues and adipose tissues where increased sugar uptake by reacting to insulin is facilitated, GLUT4 that is activated by reacting to insulin stimulation is organ-specifically expressed. GLUT4 is hardly exposed on a cell membrane in the absence of insulin stimulation, but is presented in a state of being incorporated in a vesicle group within a cell.
On the other hand, insulin stimulation can increase the amount of a GLUT4 protein exposed on a surface of a cell membrane by facilitating the transportation of the GLUT4 that is present in this intra-cellular vesicle to the cell membrane. In other words, the increased sugar uptake by insulin is achieved by insulin-dependent translocation of this GLUT4 protein to the cell membrane (membrane translocation of GLUT4). This membrane translocation of the GLUT4 in an insulin-dependent manner is a unique characteristic of the GLUT4 protein that is not observed in other GLUT families.
For the measurement of insulin-dependent sugar uptake activity in muscles, a method of measuring how sugar uptake changes by insulin in the entire muscle has been used with an uptake amount of radioisotope-labeled 2-deoxyglucose as an indicator. However, this method has a disadvantage in that an increased uptake amount of sugar purely reacting to insulin is not easily measured if sugar uptake in a ground state that is occasionally observed in tissues/organs with high basic energy consumption such as the muscles, in other words sugar uptake activity in an insulin-independent manner, is high (Biochem. Pharmacol., 2003, Vol. 65, page 249-257; and Am. J. Physiol. (Endoclinol Metab), 2002, Vol. 283, page E514-524).
Thus, the most suitable method for more accurate measurement of insulin-dependent sugar uptake activity is to accurately measure the amount of insulin-dependent translocation of an GLUT4 to a cell membrane. A method of tracing intra-cellular altered localization of an intrinsic GLUT4 using a technique such as the western blot method, etc., can be used as well. However, this method has an issue in that an advanced operation that takes time to fractionate a cell extract is needed.
As described above, it is not easy to accurately measure the membrane translocation amount of GLUT4 only, and thus, in a cultured adipose cell strain (3T3L1) with an excellent cultured cell system established, a technique for the measurement of the membrane translocation amount of GLUT4 has been developed by creating a GLUT4 with various types of tags biogenetically transfected be expressed exogenously, etc. (J. Biol. Chem., 2001, Vol. 276 (No. 45), page 42436-42444).
Furthermore, research exists in which sugar uptake promoting activity was examined by differentiating myoblast cells C2C12 expressing a GLUT4 with myc tags and treating them with a drug (European J. Pharmacol., 2000, Vol. 410, page 1-5).
However, for differentiation-type myotube cells formed by the fusion of a plurality of myoblast cells, gene transfection is difficult due to the following reasons, and an excellent cultured muscle cell system in which membrane translocation of GLUT4 reacted to insulin has not existed. These reasons include: (1) myotube cells formed by the fusion of a number of myoblast cells and which are highly differentiated cannot be fully adhered after being separated from a culture dish, and hence, gene transfection by an electroporation method often used for cultured adipose cells that are dispersed once cannot be carried out; and (2) gene transfection into undifferentiated myoblast cells can be carried out by various methods; however, myoblast cells transfected with a foreign gene frequently have a decreased differential ability, and occasionally do not become differentiated enough for myotube cells that are sufficiently developed and have insulin responsiveness. Moreover, even if they become differentiated to a certain degree, expression of the foreign gene may be inhibited, and hence, expression of differentiated myotube cells is not expected.
As a heretofore known technique that carries out highly efficient gene transfection into differentiated myotube cells, a method exists that utilizes an adenovirus vector. However, the adenovirus vector can be transmitted to humans, and therefore, high proficiency of the experimenter and research facility where adenovirus vector is used are essential, making it very difficult to carry it out easily and safely. Moreover, infection of the adenovirus vector itself frequently affects the function of cells, and thus, this method may not be suitable for accurate measurement of insulin responsiveness, etc.